This invention relates generally to the detection of genetic differences for leanness among pigs and particularly use of a genetic marker in the leptin receptor gene which is indicative of the heritable trait of low fat content in pigs.
There is an increasing consumer demand for meat products having low fat content. This demand is fueled by accumulating evidence in the scientific literature that a high consumption of animal fat, especially fat with a high proportion of saturated fatty acids, represents a significant health hazard, including risk for cardiovascular disease. Other health concerns associated with high fat meats include their high content of cholesterol and the addition of relatively high amounts of salt which are added to improve the binding characteristics since salt aids in extracting the native water binding component myosin from the meat. Furthermore an increasing number of consumers find meat products containing chemical additives such as phosphates, emulsifying additives, and anti-oxidants less acceptable.
Faced with consumers who seek a healthier meat product, meat producers are continually pressed to offer cheaper and healthier products.
Genetic differences exist among individual meat producing animals as well as among breeds which can be exploited by breeding techniques to achieve animals with these desirable characteristics. Chinese breeds are known for reaching puberty at an early age and for their large litter size. American breeds are known for their greater growth rates and leanness. Thus, it would be desirable to combine the best characteristics of both types of breeds, thereby improving pork production.
Often, however heritability for desired traits is low for example heritability for litter size is around 10%-15%. Standard breeding methods which select individuals based upon phenotypic variations do take into account genetic variability or complex gene interactions which exist. Therefore, there is a need for an approach that deals with selection for reproduction at the cellular or DNA level. This method will provide a method for genetically evaluating animals to enable breeders to more accurately select those animals which not only phenotypically express desirable traits but those which express favorable underlying genetic criteria. This has largely been accomplished to date by marker assisted selection.
RFLP analysis has been used by several groups to study pig DNA. Jung et al., Theor. Appl. Genet., 77:271-274 (1989), incorporated herein by reference, discloses the use of RFLP techniques to show genetic variability between two pig breeds. Polymorphism was demonstrated for swine leukocyte antigen (SLA) Class I genes in these breeds. Hoganson et al., Abstract for Annual Meeting of Midwestern Section of the American Society of Animal Science, Mar. 26-28, 1990, incorporated herein by reference, reports on the polymorphism of swine major histocompatibility complex (MHC) genes for Chinese pigs, also demonstrated by RFLP analysis. Jung et al. Animal Genetics, 26:79-91 (1989), incorporated herein by reference, reports on RFLP analysis of SLA Class I genes in certain boars. The authors state that the results suggest that there may be an association between swine SLA/MHC Class I genes and production and performance traits. They further state that the use of SLA Class I restriction fragments, as genetic markers, may have potential in the future for improving pig growth performance.
The ability to follow a specific favorable genetic allele involves the identification of a DNA molecular marker for a major effect gene. The marker may be linked to a single gene with a major effect or linked to a number of genes with additive effects. DNA markers have several advantages; segregation is easy to measure and is unambiguous, and DNA markers are co-dominant, i.e., heterozygous and homozygous animals can be distinctively identified. Once a marker system is established selection decisions could be made very easily, since DNA markers can be assayed any time after a blood sample can be collected from the individual infant animal.
The use of genetic differences in receptor genes has become a valuable marker system for selection. For example U.S. Pat. Nos. 5,550,024 and 5,374,526 to Rothschild et. al. disclose a polymorphism in the pig estrogen receptor gene which is associated with larger litter size, the disclosure of which is incorporated herein by reference. U.S. application Ser. No. 08/812,208 filed Mar. 6, 1997 now U.S. Pat. No. 5,935,784 filed Aug. 10, 1999 discloses polymorphic markers in the pig prolactin receptor gene which are associated with larger litter size and overall reproductive efficiency.
The present invention provides a genetic marker, based upon the discovery of a polymorphism in the leptin receptor gene, which relates to leanness in pigs. This will permit genetic typing of pigs for their leptin receptor genes and for determination of the relationship of specific RFLPs to leanness. It will also permit the identification of individual males and females that carry the gene for leanness. Thus, the markers may be selection tools in breeding programs to develop lines and breeds that produce litters containing offspring with less fat content.
The murine autosomal recessive mutations obese (ob), diabetes (db) and fatty (fa) were first reported in the 1960s. The phenotypes of animals homozygous for these mutations include severe, early-onset obesity, insulin resistance and susceptibility to diabetes. The ob gene has recently been cloned in human and mouse and its protein product identified as leptin. Subsequent research led to the identification of a receptor for leptin in mice (OBR). The gene for OB-R was shown to map to within a 5.1 cM interval of mouse Chr 4 which contains the db locus. This report was followed by two studies providing evidence that db was the gene encoding OB-R. A recent report by Chua and associates has confirmed that db, fa and OB-R are the same gene. The mouse leptin receptor gene has now been assigned the symbol, Lepr, which replaces the previously used symbols OB-R and Obr. Mapping of human leptin receptor gene (LEPR) has also recently been reported.
According to the invention a polymorphism was identified in the leptin receptor gene which is associated with the leaner phenotype typically seen in American breeds.
It is an object of the invention to provide a method of screening pigs to determine those more likely to produce offspring with low fat content.
Another object of the invention is to provide a method for identifying genetic markers for pig leanness.
A further object of the invention is to provide genetic markers for selection and breeding to obtain pigs that will be expected to have a lower fat content as exemplified by the American breeds.
Yet another object of the invention is to provide a kit for evaluating a sample of pig DNA for specific genetic markers of leanness.
Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The objects and advantages of the invention will be attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
To achieve the objects and in accordance with the purpose of the invention, as embodied and broadly described herein, the present invention provides a method for screening pigs to determine those which will be likely to produce offspring with a lower fat content when bred or to select against pigs which have alleles indicating higher fat content. As used herein xe2x80x9clow fat content or leannessxe2x80x9d means an increase in leanness above the mean of a given population. Thus, the present invention provides a method for screening pigs to determine those more likely to produce low fat offspring, and/or those less likely to produce obese offspring, which method comprises the steps 1) obtaining a sample of genomic DNA from a pig; and 2) analyzing the genomic DNA obtained in 1) to determine which leptin allele(s) is/are present. Briefly, a sample of genetic material is obtained from a pig, and the sample is analyzed to determine the presence or absence of a polymorphism in the leptin receptor gene that is correlated with leanness.
In one embodiment the polymorphism is a restriction fragment length polymorphism and the assay comprises identifying the pig leptin receptor gene from isolated pig genetic material; exposing the gene to a restriction enzyme that yields restriction fragments of the gene of varying length; separating the restriction fragments to form a restriction pattern, such as by electrophoresis or HPLC separation; and comparing the resulting restriction fragment pattern from a pig leptin receptor gene that is either known to have or not to have the desired marker. If a pig tests positive for the marker, such pig can be considered for inclusion in the breeding program. If the pig does not test positive for the marker genotype the pig can be culled from the group and otherwise used.
In a most preferred embodiment the gene is isolated by the use of primers and DNA polymerase to amplify a specific region of the gene which contains the polymorphism. Next the amplified region is digested with a restriction enzyme and fragments are again separated. Visualization of the RFLP pattern is by simple staining of the fragments, or by labeling the primers or the nucleoside triphosphates used in amplification.
It is also possible to establish linkage between specific alleles of alternative DNA markers and alleles of DNA markers known to be associated with a particular gene (e.g. the leptin receptor gene discussed herein), which have previously been shown to be associated with a particular trait. Thus, in the present situation, taking the leptin receptor gene, it would be possible, at least in the short term, to select for pigs likely to produce lean litters, or alternatively against pigs likely to produce obese or high fat litters, indirectly, by selecting for certain alleles of the leptin receptor associated marker through the selection of specific alleles of alternative chromosome 6 markers. According to the invention, examples of markers on the published PiGMaP chromosome 6 map which are linked to the leptin receptor gene include S0059, S0228, S0003, S0299, S0121, SO146 and S0031. A multiple point analysis produced the best map order of these marker and LEPR (with distance in Kosambi cM): S0059-13.3-S0228-1.0-S0003-4.4-S0299-4.5-S0121-7.9-LEPR-22.1-S0146-3.3-S0031.
The invention further comprises a kit for evaluating a sample of pig DNA for the presence in pig genetic material of a desired genetic marker located in the pig leptin receptor gene indicative of the heritable trait of low body fat. At a minimum, the kit is a container with one or more reagents that identify a polymorphism either in or associated with the pig leptin receptor gene. Preferably, the reagent is a set of oligonucleotide primers capable of amplifying a fragment of the pig leptin receptor gene that contains the polymorphism. The kit further may contain a restriction enzyme that cleaves the pig leptin receptor gene in at least one place. In a most preferred embodiment the restriction enzyme is HinfI or MboI or one which cuts at the same recognition sites.